Suppression of gene expression may be accomplished by constructs that trigger post-transcriptional or transcriptional gene silencing. These silencing mechanisms may downregulate desired polynucleotide or gene expression by chromatin modification, RNA cleavage, translational repression, or via hitherto unknown mechanisms. See Meister G. and Tuschl T., Nature, vol. 431, pp. 343-349, 2004.
A construct that is typically used in this regard contains a desired polynucleotide, which shares sequence identity with at least part of a target gene that is operably linked to a promoter and a terminator. As is well appreciated, the promoter initiates transcription, while the terminator ends transcription at a specific site and subsequently mediates polyadenylation. Such transcript processing is important for stability of the transcript and its transport from the nucleus and into the cytoplasm.
In this regard, the terminator plays an important role in conventional gene silencing constructs. For instance, WO 99/53050 describes a construct that comprises a promoter, a polynucleotide comprising a first sequence with homology to a target gene and a second sequence that is inverse complementary to the target gene, and a terminator. A terminator of conventional constructs does not necessarily have to be positioned immediately downstream from the desired polynucleotide. For instance, Mette and co-workers described a plasmid that contains a desired polynucleotide that is separated from an operably linked terminator by a hygromycin gene (Mette et al., EMBO J 18: 241-8, 1999; Mette et al., EMBO J 19: 5194-201, 2000).
Other conventional constructs designed to silence genes contain a polynucleotide in the sense or antisense orientation between promoter and terminator. Such a conventional gene silencing construct typically produces RNA transcripts that are similar in size, determined by the distance from transcription start to termination cleavage site and the poly-adenylated tail.
The present invention relates to new strategies and constructs for gene silencing that are generally more effective than conventional constructs. Furthermore, the present invention relates to new strategies and constructs for gene silencing using a polynucleotide that is not operably linked to a promoter and a terminator but is instead operably linked to two convergently-oriented promoters.